Method for expansion of epithelial stem cells

ABSTRACT

Transplantation of epithelial stem cells, cultured ex vivo on specifically treated amniotic membrane, yields, with that amniotic membrane, a surgical graft having expanded epithelial stem cells. The method of creating this graft and the graft itself provide simple and effective means to reconstruct damaged tissue, a preferred example being corneal tissue. The source of the epithelial stem cells can be a very small explant from healthy autologous and allogeneic tissue biopsy. The amniotic membrane is treated such that its extracellular matrix is maintained, but its cells are killed.

BACKGROUND OF THE INVENTION

[0001]1. Field of the Invention

[0002] This invention concerns epithelial stem cell and limbal stem celldeficiency and, more specifically, a method and graft for treating thisproblem of epithelial stem cell deficiency in, for example, thereconstruction of corneal surface.

PRIOR ART

[0003] With respect to the eye, the normal ocular surface is covered bycorneal, limbal and conjunctival epithelia. Their distinct cellularphenotypes, together with a stable preocular tear film, maintain theocular surface integrity. Severe limbal epithelial damage due to:chemical or thermal burns, Stevens-Johnson syndrome, ocular cicatricialpemphigoid, multiple surgeries and cryotherapies at the limbal region,contact lens wears and severe microbial infection can lead to limbal andepithelial stem cell deficiency. Limbal epithelial stem cell deficiencyusually is manifested with conjuctivalization, vascularization, chronicinflammation and fibrous ingrowth onto the corneal surface and cornealopacification.

[0004] When limbal deficiency is unilateral or bilateral withasymmetrical involvement, autologous limbal tissue transplantation issuggested. One major concern of autologous limbal transplantation isthat one or two limbal grafts, spanning the area of two to three clockhours of the limbus, have to be removed from the healthy fellow eye.There has been one report describing the potential complication in donoreyes. Experiments with rabbits also have shown that limbal deficiencycan occur if the central corneal epithelium is removed subsequently fromdonor eyes, with prior limbal removal. Pellegrini et al., Lancet 1997;349:990-993, reported the transplantation of corneal epithelial cellsheets expanded on 3T3 fibroblast feeder layer, for corneal surfacereconstruction in two total limbal deficiency patients.

[0005] Recently, transplantation of amniotic membrane, as a substratereplacement, has been shown by Kim and Tseng, Cornea 1995; 15:473-84, tobe effective in reconstructing the corneal surface in rabbits with totallimbal epithelial stem cell deficiency.

SUMMARY OF THE INVENTION

[0006] In one example of this invention, limbal stem cells andepitheleal cells from a small limbal biopsy, taken from a healthy eye,and in culture are expanded on a specially treated amniotic membrane.Employing such amniotic membrane as a substrate helps restore anon-inflamed limbal stroma and expand the limbal stem and epithelealstem cell populations. The resulting “product” can be transplanted, as agraft, to a denuded corneal surface, following superficial heretectomyto remove fibrovascular ingrowth. For damaged body areas other than theeye, a biopsy of healthy epithelial cells is from an adjacent tissuearea, having the same or similar biologic/histologic characteristics,i.e. histocompatible with the damaged area.

DESCRIPTION OF A PREFERRED EMBODIMENT

[0007] Limbal biopsy is performed on a healthy eye, which can be afellow eye of a patient or from another living individual. The eye lidis sterilized with Betadine® (prvidone-iodine). Under sterileconditions, 1 to 2 mm² of the limbal tissue, containing epithelial cellsand part of the corneal stroma tissue, is separated from the limbalmargin and excised from superficial corneal stroma by lamellarkeratectomy, with No. 66 Beaver® blade (Becton Dickinson, FranklinLakes, N.J.). The tissue is placed in a 35 mm dish containing 1.5 ml ofculture medium, having per ml: DMEM (Dulbecco's modified Eagle's medium)and Ham's F12 (1:1 ratio), supplemented with 0.5% DMSO (dimethylsulfoxide), 2 ug mouse EGF (epidermal growth factor), 1 ug bovineinsulin, 0.1 ug cholera toxin and 5% fetal bovine serum and it is sentimmediately to the laboratory for culture in a sterile, laminar flowhood.

[0008] Amniotic membrane (obtained from Bio Tissue, Miami, Fla.) is usedas a, culture system and is obtained, processed and preserved asreported by Tseng SCG in Am. J. Opthalmol 1997; 124:765-774 and TsengU.S. Pat. No. 6,452,142, the teaching of which is incorporated herein.The amniotic membrane, with basement membrane side up, is affixedsmoothly onto a culture plate and placed at 37° C. under 5% CO₂ and 95%air, in a humidified incubator overnight before use. Limbal explantculture is performed as previously described (Tsai and Tseng, InvestOpthalmol Vis Sci 1988; 29:97-108; and Tsai, et al., Invest OpthalmolVis Sci 1994; 35:3865-2875), with some modifications. Instead oftransfer onto a plastic substrate, as taught in these references, thelimbal explant with the epithelial stem cells is planted/transferredonto the basement membrane side of the amniotic membrane in a 35 mm dishcontaining 1 ml of the above described culture medium. The medium ischanged every two days, and the culture is maintained for 2 to 3 weeks,by which time the epithelial stem cells have grown and spread to form acell layer covering an area of about 2 to 3 cm in diameter, for cornealsurface reconstruction. For other tissue repair, more or less epithelialstem cell layer area can be employed, as needed.

[0009] Continuing with the example of corneal repair, followingperiotomy at the limbus, the perilimbal subconjunctival scar andinflamed tissues are removed to the bare sclera. The fibrovasculartissue of cornea is removed by lamellar keratectomy, with No. 57 and 66Beaver blades, in a manner similar to that described for allograftlimbal transplantation (Tsai and Tseng Cornea 1994; 13:389-400). Forthose patients with partial to total limbal corneal damage, but withnormal central cornea, the cultivated limbal epithelial stem cells withthe amniotic membrane is used as a sectorial limbal corneal graft, or alimbal equivalent, fashioned according to the size of the recipient eye,and transplanted to the corresponding recipient limbal area (from 90° to360°). For those patients with total limbal and corneal surface damage,the novel graft is used as a whole lamellar corneal tissue, or a limbalcorneal equivalent, and transplanted as lamellar keratoplasty to coverthe entire area.

[0010] A properly sized, cultured, epithelial stem cell sheet on thetreated amniotic membrane substrate is affixed to cover the entiredefect with the epithelial side up, which can be readily identified byfluorescent staining or by the presence of loosely attached originalexplant. The graft then is secured to the damaged site. For a damagedcornea, securing can be by interrupted 10-0 nylon sutures on the cornealside, and interrupted 8-0 Vicryl® sutures to the surroundingconjunctival edge with episcleral anchorage. During the entireprocedure, the cultured epithelium stem cell layer is protected fromexposure, drying and abrasion by a coating of sodium hyaluronic(hyaluronate) acid Healon® (Pharmacia & Upjohn AB, Uppsala, Sweden). Theoriginal explant tissue can be removed from the amniotic membrane at theend of surgery. If this graft is over the cornea, the eye is pressurepatched overnight, a therapeutic contact lens is placed the next day,for one week; and topical prednisolone acetate 1% solution isadministered four times a day for the first week, twice a day for thenext two weeks, and followed by 0.1% fluorometholone twice a day for 2to 3 months, depending on the severity of conjunctival inflammationaround the surgical area.

[0011] As stated hereinabove, the explant is cultured onto the basementmembrane side of the specially treated amniotic membrane. After 2 to 3weeks, the epithelial stem cells grow to form a sheet approximately 2 to3 cm² in size on the amniotic membrane. Flat-mount preparation shows theepithelial stem cell layer is negative to PAS and Alcian blue staining;and the bare amniotic membrane is stained purple. Histologicalexaminations show that epithelial sheet is composed of 4 to 5 stem celllayers at the margin of the sheet and from 1 to 4 cell layers in thearea between the margin and original explant tissue. Ultrastructuralexaminations reveal the presence of loose and wide intercellular spacesand basement membrane structure, with focal condensation ofelectron-dense ground substance at the basal cell-amniotic membranejunction.

[0012] With respect to an embodiment of the invention specific to thetreatment of eyes, a mean (±SD) follow-up period of 14.8±1.9 monthsshows varying degrees of visual improvement based on the Snellen visualacuity scale. All eyes show complete re-epithelialization in 2 to 4 dayswith a mean (±SD) period of 2.7±0.8 days. The reconstructed cornealsurfaces show reduced inflammation and regression of vascularizationwithin 1 to 2 weeks. One month after operation, the corneal clarity isimproved and the surface smooth and wettable.

[0013] Depending on the area of limbal corneal damage, the culturedepithelial stem cells, expanded on a specially prepared amnioticmembrane substrate, can be used as a limbal equivalent or alimbal-corneal equivalent.

[0014] Limbal deficiency of the donor eye, due to removal of arelatively large piece of limbus for transplantation, has been reportedin rabbits. Thus, the new method and resulting graft of this inventionsubstantially reduces the potential complications to the donor eye,since only a small piece of limbus is removed. Moreover, this methodalso can be performed in eyes with bilateral limbal deficiency withasymmetrical involvement. Ex Vivo expansion of autologous epithelialstem cells on specially pre-treated amniotic membrane providessufficient epithelial stem cells for transplantation in 2 to 3 weeks.For patients with bilateral total limbal deficiency, limbus fromcompatible sources, another living, related individual, should beconsidered for use according to this invention.

[0015] The use of uniquely pre-treated amniotic membrane basedautologous epithelial stem cells for transplantation also provides allthe beneficial effects inherent in amniotic membrane transplantation,including facilitating epithelization, reducing inflammation andscarring, and substrate replacement when the underlying stromal tissueis destroyed. Most importantly, amniotic membrane, pre-treated accordingto Tseng, provides a natural substrate for epithelial stem cells to bepreserved and expand, forming the autologous cell mass necessary forcorneal reconstruction. Moreover, because only the autologous cells aretransplanted, immunosuppression is not required after transplantation.For allogeneic stem cells transplanted in this manner, the rejectionrate may be lessened, since only epithelial stem cells, without othercell types, are transplanted.

[0016] As stated hereinabove, this unique graft and the method of itsforming, by use of specially treated amniotic membrane, upon whichepithelial stem cells from an explant are expanded, have uses beyondthat of eye surgery, for example, repair of burned skin areas;especially when the donor site of the explant needs to be small.Likewise, the biopsy, from which the small explant is obtained, need notbe of limbal tissue. The explant is to have healthy tissue, containepithelial stem cells and be histocompatible with the recipient site forthe graft. If the biopsy cannot be from the same body part as therecipient site, a corresponding similar body part can be chosen for theexplant; just as in the preferred example of a damaged eye being therecipient site and the other eye—the healthy eye—providing donorexplant.

[0017] It is believed that a unique and inventive surgical graft andmethod of its creation have been disclosed sufficiently for thoseskilled in the art to practice the invention without significantexperimentation, as well as develop modifications which lie within thespirit and scope of the invention as defined by its claims.

What is claimed is:
 1. A surgical graft to be applied to a recipientsite, said graft comprising: an amniotic membrane; and epithelial stemcells expanded on said amniotic membrane.
 2. A surgical graft accordingto claim 1 in which, said amniotic membrane has a basement membraneside; and said epithelial stem cells are expanded on said basementmembrane side.
 3. A surgical graft according to claim 1 in which, saidamniotic membrane has a stroma side which is the side of said graft tobe applied to the recipient site.
 4. A surgical graft according to claim1 in which, limbal tissue, from a limbal biopsy performed on a healthyeye, is the source of said epithelial stem cells.
 5. A surgical graftaccording to claim 4 in which, said limbal tissue is autologous.
 6. Asurgical graft according to claim 4 in which, said limbal tissue isheterologous.
 7. A surgical graft according to claim 6 in which, saidlimbal tissue is from a cadaver.
 8. A surgical graft according to claim4 in which, said limbal tissue approximates 1-2 mm2 in surface area. 9.A surgical graft according to claim 1 in which, said amniotic membranehas a basement membrane side; and said epithelial stem cells arecultured ex vivo on said basement membrane side of said amnioticmembrane.
 10. A surgical graft according to claim 9 in which, saidepithelial stem cells originate from tissue treated in a culture mediumand then transferred to said amniotic membrane.
 11. A surgical graftaccording to claim 10 in which, said amniotic membrane has cells and anextracellular matrix having integrity; and prior to said ex vivoculture, said cells of said membrane are killed, but the integrity ofsaid extracellular matrix is maintained.
 12. A surgical graft accordingto claim 11 in which, said expanded epithelial stem cells have as theirorigin limbal explant tissue from a healthy eye.
 13. A surgical graftaccording to claim 12 in which the recipient site is an eye; and saidlimbal explant tissue is not required as a component of said graft,after said graft is applied to the recipient site.
 14. A surgical graftaccording to claim 12 in which, said graft is a sectorial limbal cornealgraft for a recipient site having partial to total limbal cornealdamage, with normal central cornea.
 15. A surgical graft according toclaim 12 in which, said graft defines a whole lamellar corneal tissuefor a recipient site having total limbal and corneal surface damage. 16.A surgical graft according to claim 1 in which, the source of saidepithelial stem cells is tissue from a biopsy taken from a healthy sitecorresponding biologically and histocompatible to the recipient site.17. A method for creating a surgical graft for a damaged recipient site,said method comprising the steps of: a) obtaining a biopsy from ahealthy donor site, said biopsy containing epithelial stem cells; b)placing, as an explant, said biopsy onto an amniotic membrane; and c)enabling said epithelial stem cells to expand on said amniotic membrane.18. A method according to claim 17 in which said amniotic membranehaving a basement membrane side; and said step of placing includingmounting said explant onto said basement membrane side.
 19. A methodaccording to claim 18 in which, said mounting causing said expandedepithelial stem cells to be positioned face up with reference to thedamaged recipient site.
 20. A method according to claim 17 in which saidstep of obtaining includes; performing a limbal biopsy on a healthy eye.21. A method according to claim 20 in which, said step of performing ison an allogeneic eye.
 22. A method according to claim 17 in which, saidstep of obtaining a biopsy is from a histocompatible donor site.
 23. Amethod according to claim 17 in which said step of enabling includes;culturing said explant in a culture medium, prior to said step ofplacing.
 24. A method according to claim 23 in which, said step ofculturing is ex vivo.
 25. A method according to claim 17 in which saidamniotic membrane has cells and an extracellular matrix; and said stepof enabling includes, prior to said step of placing; killing said cellsof said amniotic membrane, but maintaining its extracellular matrix. 26.A method according to claim 17 in which said step of enabling including;culturing said amniotic membrane, with its explant, for a durationsufficient for said epithelial stem cells to have expanded to an area ofabout 2 to 3 cm in diameter.
 27. A method according to claim 26 in whichsaid culturing including; employing a medium which is changedapproximately every two days for upwards to three weeks.
 28. A methodaccording to claim 17 including; removing said explant from saidamniotic membrane after securing said graft surgically to the recipientsite.